Nonribsomal peptide (NRP) natural products biosynthesized by nonribosomal peptide synthetases (NRPSs) are among the most important therapeutics known to mankind, including penicillin, vancomycin and cyclosporin. Newly approved lipo-NRPs such as daptomycin and anidulafungin (a semisynthetic derivative of echinocandin B, the target of this proposal) are important weapons in combating bacterial and fungal infections, respectively. Filamentous fungi are prolific producers of NRPs and represent an important source for future natural-product based drug discovery. Recent genome sequencing of >30 fungi species have revealed each genome encodes far more NRPS than the identified NRPs, with a majority of the NRPS genes being silent during laboratory culturing conditions. Therefore, having the abilities to predict NRP product structure based on sequence, to activate otherwise cryptic pathways in both native and heterologous hosts, and to manipulate the NRPSs towards the biosynthesis of targeted derivatives are important goals towards realizing the fungal biosynthetic potential. Our proposed work here represents the first comprehensive study of a fungal NRPS to date. We will focus on the biosynthesis of echinocandin B, which is a fungal lipopeptide that contains six highly hydroxylated amino acids. We have recently identified the gene cluster from the producing organism Aspergillus nidulans sp. This collaborative proposal between biosynthetic (Tang) and a synthetic (Garg) labs aims to address the unique chemistry and biology associated with fungal NRPSs. Our proposed work is based on extensive preliminary results that have generated deep knowledge with the fungal systems, as well genetic and biochemical tools. Using a combination of genetic knockout/knock-in, heterologous expression in Saccharomyces cerevisiae and in vitro biochemical interrogation, we will dissect the echinocandin NRPS (EcdA) and associated enzymes using the following four aims: 1) Examine the activation and biosynthesis of unusual amino acids; 2) Characterization of the hydroxylases in the echinocandin pathway; 3) Heterologous reconstitution of fungal NRPS; and 4) Genome mining of cryptic fungal NRPSs.